Anaplasma phagocytophilum (A. phagocytophilum) is the aetiological organism of tick-borne fever in cattle and sheep, and granulocytic anaplasmosis in dogs and human. Reports of livestock and wildlife infection and associated zoonotic risk of human infection with A. phagocytophilum have been reported across the world. A previous study has reported two strains, also called ecotypes, in ticks collected from across England and Wales. This study examined A. phagocytophilum strains present in livestock and wildlife in Great Britain (GBR), with a particular focus on cattle. Clinical submissions (EDTA blood) from livestock were received by APHA for tick-borne disease testing. Of these 24 (cattle: n = 21; sheep: n = 3) were confirmed to be infected with A. phagocytophilum using a PCR targeting the msp2 gene. Further submissions from roe deer (n = 2), red deer (n = 2) and Ixodes ricinus ticks (n = 22) were also shown to be infected with A. phagocytophilum. Subsequent analysis using a nested PCR targeting the groEL gene and sequencing, confirmed the presence of ecotype I in cattle, sheep, red deer and Ixodes ricinus, and ecotype II in roe deer and I. ricinus ticks removed from the deer carcass. Despite the presence of two ecotypes, widely distributed in ticks from England and Wales, only ecotype I was detected in cattle in this study.